Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep;12(9):e1054.
doi: 10.1002/ctm2.1054.

Loss of Sirtuin 1 (SIRT1) potentiates endothelial dysfunction via impaired glycolysis during infectious challenge

Ryan J Stark  1 , Stephen R Koch  1 , Cody L Stothers  1 , Allison Pourquoi  1 , Celia K Lamb  1 , Michael R Miller  1 , Hyehun Choi  1
Affiliations

Loss of Sirtuin 1 (SIRT1) potentiates endothelial dysfunction via impaired glycolysis during infectious challenge

Ryan J Stark et al. Clin Transl Med. 2022 Sep.
No abstract available

PubMed Disclaimer

Conflict of interest statement

None declared.

Figures

FIGURE 1
FIGURE 1
(A) SIRT1 mRNA relative values on day 0 (<24 h) of hospitalisation are shown for children (top, ≤10 years of age) or adult (bottom, >18 years of age) patients with or without sepsis from the respective GEO Datasets (GSE26378, children and GSE134364, adults). The median expression value is shown with a horizontal bar per condition with 75% interquartile range normalised to the respective controls (children: controls n = 21, sepsis n = 79; adults: controls n = 83, sepsis n = 156). (B) qRT‐PCR values of SIRT1 mRNA normalised to GAPDH control (top, n = 4 individual replicates) or SIRT1 protein expression with associated western blots normalised to tubulin (bottom, n = 6 individual replicates) either with vehicle or LPS treatment (100 ng/ml) for 6 or 24 h in HMVECs. (C) Top: Transendothelial electrical resistance (TEER) of HMVECs treated with scrambled siRNA (siControl) or siRNA to SIRT1 for 72 h prior to LPS exposure of 100 ng/ml and subsequently followed for 24 h. Data are normalised to baseline prior to LPS exposure. Open circles represent HMVECs exposed to siControl for 72 h without LPS (vehicle). Bottom: Area under the curve (AUC) relative to change from baseline is shown for the TEER. †p < .05 between indicated group and control. *p < .05 between designated groups. n = 4–6 individual replicates per group. (D) Top: TEER of HMVECs pretreated with 0.1 μM EX527 30 min prior to LPS compared to vehicle control. All data are normalised to baseline resistance per condition prior to addition of EX527. Bottom: AUC relative to change from baseline resistance over time calculated for all conditions following EX527 or vehicle addition out to 24 h. †p < .05 between indicated group and control. *p < .05 between designated groups. n = 3 individual replicates per group. (E) Relaxation of isolated murine mesenteric arteries to escalating doses of acetylcholine (ACh, top) or sodium nitroprusside (SNP, bottom) treated with control (DMSO, n = 8), EX527 (0.1 μM, n = 8), LPS (100 ng/ml, n = 7) or EX527 with LPS (n = 8) for 24 h prior to myography assessments. †p < .05 between LPS and EX527 + LPS at the respective dose. #p < .05 for EC50 between LPS and EX527 + LPS. ‡p < .05 for EC50 between EX527 and EX527 + LPS
FIGURE 2
FIGURE 2
(A) eNOS, FOXO1 and TFAM mRNA transcript relative values are shown for children with or without sepsis from the respective GEO Dataset (GSE26378). The median expression value is shown with a horizontal bar per condition with 75% interquartile range normalised to the respective cohort controls. (B) Expression values for eNOS, FOXO1 or TFAM in comparison to individually matched SIRT1 transcript values for control patients or patients with sepsis. Associated coefficients of determination (R 2) are shown with Spearman r correlation coefficient p values. Linear regression (solid lines) with 95% CI (dotted lines) are displayed. *p < .05 between indicated group and control cohort. NS = non‐significant. (C) Densitometry analysis and associated images of whole cell lysate western blots (n = 6 individual replicates per group) calculated for SIRT1, eNOS, FOXO1 or TFAM expression relative to tubulin. HMVECs were treated with respective siRNA for 72 h prior to LPS exposure (100 ng/ml) at the times indicated. (D) Proximity ligation assay (PLA) counts normalised to cell number for SIRT1 interactions with eNOS, FOXO1 and TFAM with or without LPS exposure for 6 h. Associated representative confocal images (40×, line bar represents a distance of 50 μm) are shown for SIRT1‐eNOS (cyan), SIRT1‐FOXO1 (purple) and SIRT1‐TFAM (red) PLA reactions. n = 5 individual replicates per group. *p < .05 between designated groups. †p < .05 between indicated group and respective siRNA control. NS = non‐significant
FIGURE 3
FIGURE 3
(A) Seahorse XFe96 assays for oxygen consumption rate (OCR, left) or extracellular acidification rate (ECAR, right) using Control and SIRT1 siRNA treated HMVECs with 6 h of LPS exposure (100 ng/ml) prior to assays. (B) Respective components of OCR via calculation as follows: ‘Basal OCR’ = pre Oligomycin – post Antimycin/Rotenone, ‘ATP‐linked’ = pre Oligomycin – post Oligomycin, ‘Max Respiration’ = post FCCP – post Antimycin/Rotenone, ‘Reserve OCR’ = post FCCP – pre FCCP. (C) Respective ECAR components with calculations as follows: ‘Basal ECAR’ = pre Glucose – post 2‐deoxyglucose (2‐DG), ‘Glycolysis’ = post Glucose – pre Glucose, ‘Max Glycolysis’ = post Oligomycin – post 2‐DG, ‘Reserve ECAR’ = post Oligomycin – pre Oligomycin. n = 11‐12 individual replicates per group. (D) Seahorse XFe96 assays for oxygen consumption rate (OCR, left) or extracellular acidification rate (ECAR, right) using Control and SIRT1 siRNA treated HMVECs with 24 h of LPS exposure or condition media prior to assays. Components of OCR (E) or ECAR (F) for respective siRNA ECs treated with or without 24 h of LPS exposure. *p < .05 between designated groups. †p < .05 between indicated group and respective siRNA control
FIGURE 4
FIGURE 4
(A) Hexokinase 2 (HK2), phosphofructokinase (platelet, PFKP) or pyruvate kinase M2 (PKM2) mRNA transcript relative values are shown for children with or without sepsis from the respective GEO Dataset (GSE26378). The median expression value is shown with a horizontal bar per condition with 75% interquartile range normalised to the respective cohort controls. (B) Expression values for HK2, PFKP or PKM2 in relation to individually matched transcript levels for SIRT1, eNOS, FOXO1 or TFAM for patients with sepsis. Associated coefficients of determination (R 2) are shown with Spearman r correlation coefficient p values. Linear regression (solid lines) with 95% CI (dotted lines) are displayed. *p < .05 between indicated group and control cohort. NS = non‐significant. (C) Densitometry analysis (left) and associated images (right) of whole cell lysate western blots (n = 6 individual replicates per group) calculated for HK2, PFKP or PKM2 expression relative to tubulin. HMVECs were treated with respective siRNA for 72 h prior to LPS exposure (100 ng/ml) at the times indicated. *p < .05 between designated groups. †p < .05 between indicated group and respective siRNA control. NS = non‐significant
See this image and copyright information in PMC

References

    1. Boeddha NP, Schlapbach LJ, Driessen GJ, et al. Mortality and morbidity in community‐acquired sepsis in European pediatric intensive care units: a prospective cohort study from the European Childhood Life‐threatening Infectious Disease Study (EUCLIDS). Crit Care. 2018;22:143. - PMC - PubMed
    1. Shankar‐Hari M, Harrison DA, Rubenfeld GD, Rowan K. Epidemiology of sepsis and septic shock in critical care units: comparison between sepsis‐2 and sepsis‐3 populations using a national critical care database. Br J Anaesth. 2017;119:626‐636. - PubMed
    1. Wang X, Zhang Q, Yan Y, Yang Y, Shang X, Li Y. Clinical Significance of pro‐inflammatory cytokines and their correlation with disease severity and blood coagulation in septic patients with bacterial co‐infection. Shock. 2021;56:396‐402. - PubMed
    1. Stark RJ. Endothelial‐dependent responses correlate with pediatric SOFA Scores during severe sepsis and septic shock. J Cardiovasc Transl Res. 2022. Online ahead of print - PMC - PubMed
    1. Ince C, Mayeux PR, Nguyen T, et al. The endothelium in sepsis. Shock. 2016;45:259‐270. - PMC - PubMed

Publication types